JPH0247475B2 - - Google Patents

Description

[Detailed description of the invention]

The use of β-lactam antibiotics in the chemotherapy of bacterial infections
(cephalosporin)-β-lactam-amidohydrolase, EC3526] has been constrained by the emergence of β-lactam-resistant pathogens. This enzyme breaks down the C--N bond in the β-lactam ring, thereby causing the β-lactam used to lose its antibiotic properties. To ensure the desired therapeutic effect when treating bacterial infections with β-lactam antibiotics, there must be a reliable knowledge of whether the clinical isolate produces β-lactamases before starting treatment. must be maintained. If the detection of β-lactamase is positive, it is preferable to use a β-lactam antibiotic that is resistant to β-lactamase or use another clinically available antibiotic. A series of procedures have been described for the detection of β-lactamase-producing pathogens (“J.Pharmacol.”
15, pp. 81-91 (1963)]. However, all the methods presented so far are not completely satisfactory. This is because they all contain indicators (iodine/starch, hydroxyamines,
This is because it requires the addition of a pH indicator). In contrast to β-lactamase detection using test articles coupled to indicators, chromogenic β-lactamase substrates have the advantage that cleavage of the β-lactam ring by this enzyme directly produces a color change in the visible region of the spectrum. have The chromogen β-
A lactamase substrate (compound 87/312) has been described. However, the authors report that bathochromic migration of solution color of this compound occurs not only in the presence of β-lactamases, but also in serum, animal tissues, proteins, milk, cysteine, glutathione, mercaptoethanol, and 2,3 - Also appears in a non-specific manner after addition of dimercaptopropanol-1. Therefore, the use of compound 87/312 may lead to false positive determinations of clinical isolates. It was therefore an object of the present invention to develop a novel method which indicates the presence of β-lactamases by an optically well visible deepening of the color from pale yellow to intense yellow-orange and is stable towards the above-mentioned additives. Located in the chromogen cephalosporin. Detection of β-lactamase is β
- It can be carried out equally well using intact cells of a lactamase-producing pathogen or with enzymes isolated from this bacterium using standard methods. Beyond pure detection, the compounds according to the invention are also very well suited for measuring the specific activity of isolated β-lactamases. The compounds according to the invention are degraded by all β-lactamases from Gram-negative bacteria with a deepening of the color as indicated above. They are particularly useful for bacteria of clinical interest, such as E. coli P _TEM , Klebsiella aerogenes
aerogenes) 1082E, Pseudomonas aeruginosa 18SH, Enterobacter cloacae P99
and β-lactamase from Bacteroides fragilis 620. The compounds according to the invention are likewise degraded by β-lactamases from Gram-positive bacteria, such as Staphylococcus aureus R85. Detection of β-lactamase activity using the compounds of the invention is carried out directly, ie, without the use of a test article coupled to an indicator. This is because the chromophore substituents present in the above-mentioned context with the object of the invention are quantitatively removed simultaneously with the cleavage of the β-lactam ring, which directly results in the color variations mentioned above. . It is now surprising that the compounds according to the invention, as opposed to β-lactamase substrates of type 87/312,
Very susceptible to hydrolytic degradation in the temperature range -180°C to +60°C, but especially in the neutral PH range, in aqueous solutions suitable for the activity of β-lactamases or for bacterial viability. It was confirmed to be stable. Storage of the final prepared solution is preferably carried out at −20°C, where the solution is almost indefinitely stable, while neutrally buffered solutions have their usability at refrigerator temperature and even room temperature. Can be stored in a fire container for a long time without being harmed. Furthermore, the compounds according to the invention can be applied to serum, proteins, milk,
Cysteine, glutathione, mercaptoethanol and 2,3-dimercaptopropanol-1
(dimercaprol), thereby providing another unexpected advantage over β-lactamase substrates of type 87/312. Therefore, the object of the present invention is the general formula Cephalosporins have a chromogenic radical at the 3'-position. Here, in the above formula, R ¹ is hydrogen,

[Formula] or

[Formula], and X is the formula (wherein R ² represents nitro and R ³ represents hydrogen or carboxy). In the residue X having the above formula, R ² represents a nitro group, especially in the p- or o-position relative to the sulfur. Particularly preferred meanings of X are p-nitrophenylthio, o-nitrophenylthio, m-carboxy-p-nitrophenylthio and p-carboxy-o-nitrophenylthio. The subject of the invention is furthermore the formula ^A compound of the formula and if R ¹ means hydrogen, optionally

[Formula] or

This is a method for producing a cephalosporin having the general formula, which is characterized by reacting it with the formula [Formula] or a reactive derivative thereof. The starting materials necessary for carrying out the process according to the invention are known in the literature or can be prepared by methods known in the literature. The reaction of a compound with a compound of the formula H-X is preferably carried out in an aqueous medium, inter alia methanol, ethanol, isopropanol, acetonitrile, acetone, etc. in order to improve the solubility of the components participating in the reaction. Water-miscible organic solvents such as dimethylformamide, dimethylacetamide, tetrahydrofuran or dioxane may be added. The compounds of formula H-X can also be used in the form of salts, such as alkali or alkaline earth salts, especially sodium or potassium salts, or salts with optionally substituted ammonium cations. The reactants can be reacted in any proportion, but especially in molar ratios from 1 to about 5. The temperature of the reaction is not critical, but is preferably about 20-80°C. It is preferred to introduce the reaction mixture into an organic, water-soluble or water-insoluble solvent boiling at the desired reaction temperature. The reaction temperature is thereby kept constant. During the reaction, the PH value of the reaction mixture ranges from about 5 to 8, especially 6.5 to 7.5, by the addition of acid or base.
is maintained within the range of In another embodiment, the formula (wherein R ¹ represents hydrogen and X has the meaning given above)
Cephalosporin with

[Formula] or

The compounds of the present invention can be produced by reacting with the formula or activated derivatives thereof in a manner known per se. Activated derivatives include in particular halides, especially chlorides and bromides, as well as anhydrides and mixed anhydrides, azides and activated esters, especially p-nitrophenol, 2,4-dinitrophenol, methylenecyanohydrin, N- Esters with hydroxysuccinimide and N-hydroxyphthalimide, particularly preferably 1-hydroxybenzotriazole and 6-chloro-1
Esters with -H-hydroxybenzotriazole are suitable. Mixed anhydrides are particularly suitable with lower alkanoic acids, such as acetic acid, and particularly preferred are mixed anhydrides with substituted acetic acids, such as trichloroacetic acid, pivalic acid or cyanacetic acid. However, also particularly suitable are mixed anhydrides with carbonic acid half esters obtained, for example, by reaction of carboxylic acids having the formula with benzyl chloroformate, p-nitrobenzyl ester, isobutyl ester, ethyl ester or allyl ester. It is a thing. The activated derivative may be reacted as an isolated material or as such. Generally, the reaction of a cefem derivative (wherein R ¹ represents hydrogen) with a carboxylic acid or an activated derivative thereof is carried out in the presence of an inert solvent. In particular, chlorinated hydrocarbons such as methylene chloride and chloroform, ethers such as diethyl ether, diisopropyl ether and above all tetrahydrofuran and dioxane, ketones such as acetone and butanone, above all amides such as dimethylformamide and dimethylacetamide or Water is suitable. It is also preferred to use mixtures of the aforementioned solvents. This is a cefem compound (in the formula R ¹
represents hydrogen) with activated derivatives of carboxylic acids obtained in situ. Cefem compound (in the formula R ¹ represents hydrogen)
The reaction between the aforementioned carboxylic acid or its activated derivative is carried out at a temperature of about -50°C to about +80°C, especially -20°C to
It is carried out at a temperature range of +50°C, but particularly preferably -20°C to room temperature. The reaction time depends on the reactants, temperature and solvent or solvent mixture and is usually from 15 minutes to about 72 hours. The reaction of the activated derivative of the carboxylic acid with the cefem compound having the formula in which R ¹ represents hydrogen is carried out inter alia in an alkaline medium with a pH of 7 or above. This includes adding, inter alia, potassium or sodium carbonate, potassium or sodium bicarbonate, potassium or sodium hydroxide, pyridine or a trialkylamine, such as triethylamine, N-methylmorpholine, ethyldiisopropylamine, or potassium tertiary Add a base such as butyrate. The compound according to the invention having the formula H
The aqueous phase is extracted with acetic acid ester or other suitable solvent to remove unreacted starting components with -
It can be isolated by acidification to PH3, thereby precipitating out the compound and separating it, for example by filtration. Another embodiment for isolating the compounds according to the invention is to extract the aqueous phase at a pH of 2 to 4, particularly at a pH of 3, using a water-immiscible solvent such as, inter alia, acetic acid ester. After concentrating the extract under vacuum, the reaction product is precipitated by adding an organic solvent in which the reaction product is difficult to dissolve, such as ether or petroleum ether. In yet another embodiment, the reaction product has the formula It can also be extracted directly from the reaction batch at pH values higher than PH4 into water-immiscible organic solvents such as acetic acid esters, among others. The subsequent processing is performed as described above. 7-β-thien-2-yl-acetamide-3 particularly preferred for general detection of β-lactamases
-[(4-Nitro-3-carboxyphenyl)-thiomethyl]-3-cephem-4-carboxylic acid is weakly yellow colored (λ _nax 344 nm) in buffered solution (PH 7.3). The characteristic yellow color (λ _nax 405 nm) of p-nitrothiophenolate results after reaction with a β-lactamase, such as the enzyme from Enterobacter cloacae P99. The intensity of the color naturally depends on the pH value of the solution and reaches its maximum at pH 8.0. However, for normal measurement, PH
A value of 7.3 is sufficient. The most significant absorbance difference between substrate and reaction product is at 415 nm. Similar reactions of this preferred compound are also performed with other β-lactamases, such as β-lactamases from E. coli R _TEM , Klebsiella aerogenes 1082E, Pseudomonas aeruginosa 18SH, Bacteroides fragilis 620, and Staphylococcus aureus R85. Other chromogenic cephalosporins according to the invention produce a corresponding color change upon reaction with β-lactamases. The compounds according to the invention can be prepared, for example, by adding an isolated β-lactamase or a cell suspension of a β-lactamase-producing pathogen directly to a neutrally buffered aqueous solution of the chromogen cephalosporin.
By selecting an appropriate concentration of the chromogen cephalosporin used in this case, a characteristic color development is observed, which is then used for the detection of β-lactamases. Alternatively, the resulting clinical isolate in the form of a colony residue may be suitably pre-wetted with an aqueous solution of the compound according to the invention, preferably on wet test strips made of paper or other suitable carrier materials, e.g. Coated on absorbable synthetic materials, dextran or other natural polymers. Here again, β-lactamase positive samples show a characteristic color change within a short time. Furthermore, the compounds according to the invention can be used, for example, in the purification of β-lactamase in order to locate the β-lactamase peak in column chromatographic separation methods. Another preferred use of the compounds according to the invention is a suitable β-
It consists in enzyme-mechanical studies for the determination of β-lactamase inhibitors, such as clavulanic acid, which completely or partially interfere with the reaction between lactamases and the compounds according to the invention. The present invention will be explained below with reference to Examples, but these Examples are not intended to limit the present invention. Example 1 7-β-thien-2-yl-acetamide-3
-[(4-nitrophenyl)-thiomethyl]-3-
Cefem-4-carboxylic acid 2 g of p-nitrothiophenol was added to 7-thien-
4.2 g of 2-yl-acetamito-cephalosporanic acid sodium salt and 120 ml of acetone/water (1:1)
Stir for 2 hours at PH6.5-7.2 and 64°C. A further 0.5 g of p-nitrothiophenol is added and the mixture is stirred for an additional 2 hours. After cooling to room temperature, the pH was adjusted to 7.0 using 2N hydrochloric acid, extracted twice with acetate, the acetate residue was removed from the aqueous phase on a rotary evaporator, and the aqueous phase was diluted with 2N at 0 °C.
Acidify to PH2.0 using hydrochloric acid. A pale yellow precipitate forms, which is filtered off with suction and dried under vacuum with phosphorus pentoxide. 3.2 g of the title compound are obtained. NMR (DMSO- _d6 ): δ=6.9-7.5ppm (thienyl, 3H) δ=4.2ppm (s, 2H, _CH2 -CO) δ=9.0ppm (d, 1H, CONH) δ=5.6ppm (q , 1H, C-7-H) δ = 5.1ppm (d, 1H, C-6-H) δ = 3.8ppm (s, 2H, C-2-CH ₂ ) 3.6 (AB, 2H, CH ₂ -S ) δ=7.5ppm (4H, p-disubstituted-phenyl) 8.1 Example 2 7-β-phenylacetamido-3-[(4-nitro-3-carboxy-phenyl)-thiomethyl]-3-cephem-4 -Carboxylic acid 1.95 g of 7-phenylacetamide-cephalosporanic acid are dissolved in 100 ml of water under a stream of nitrogen at pH 7.0 by adding sodium bicarbonate.
This was added to 5-mercapto-2-nitro- in 12 ml of water.
A solution consisting of 6.2 mol of benzoic acid disodium salt is added dropwise. The mixture is stirred at PH 6.5-7.2 for 1 hour at room temperature and then for 4 hours at 60°C. further underwater
After addition of 3.8 mol of 5-mercapto-2-nitro-benzoic acid disodium salt, a further 5 hours 60
Stir, cool, adjust to PH5.0 and 100 °C each
Stir 3 times with ml portions of acetic ester. Remove the acetate ester residue from the aqueous phase and acidify to pH 2.0 in an ice bath. Separate the precipitate and cool at 40℃
Dry under vacuum over phosphorus pentoxide. 2.6 g of the title compound are obtained. mp98~110℃ (decomposition). NMR (DMSO- _d6 ): δ=9.1~9.0ppm (d, 1H, CONH) δ=8.1~7.0ppm (m, 3H, aromatic proton) δ=7.4ppm (s, 5H, phenyl) δ= 5.7ppm (q, 1H, C-7-H) δ = 5.2ppm (d, 1H, C-6-H) δ = 4.4ppm (s, 2H, CH ₂ -S) δ = 3.5ppm (s, 4H , _CH2CO and C-2-
_CH2 ) Example 3 7-β-thien-2-yl-acetamide-3
-[(4-nitro-3-carboxyphenyl)-
16.7 g of 7-thien-2-yl-acetamido-cephalosporanic acid sodium salt are dissolved in 650 ml of water and stirred under a stream of nitrogen. 100ml water
50 mmol of 5-mercapto-2-nitro-benzoic acid disodium salt in the solution was slowly added dropwise. At that time, the pH is maintained at 7.0. 60℃ for 6 hours at PH6.9-7.1
and then leave in the refrigerator. PH
5.0 and this solution is extracted three times with 200 ml each of acetic ester. The remaining acetate ester is removed from the aqueous phase, cooled to 0° C. and acidified to PH 2.0 using 2N hydrochloric acid. Dry the precipitate with phosphorous pentoxide. 22 g of the title compound are obtained. mp99
~104℃ (decomposition). NMR (DMSO-d ₆ ): δ = 9.0-9.2ppm (d, 1H, CONH) δ = 7.1-8.1ppm (m, aromatic proton) δ = 6.8-7.0ppm (m, 2H, thienyl 3H + 4H) δ = 5.6ppm (q, 1H, C-7-H) δ = 5.1ppm (d, 1H, C-6-H) δ = 4.3ppm (s, 2H, CH ₂ -S) δ = 4.8ppm (s, 2H, CH ₂ CO δ = 4.5 ppm (AB, 2H, C-2-CH ₂ ) Example 4 7-β-amino-3-[(4-nitro-3-carboxy-phenyl)-thiomethyl]-3- Cefem-4-carboxylic acid 2.2 g of 7-amino-cephalosporanic acid was added to 150 ml of water under a nitrogen stream using sodium bicarbonate.
Dissolve at pH7.0. 10 mmol of 5-mercapto-2-nitro-benzoic acid disodium salt in 20 ml of water are added dropwise at pH 6.8-7.3. Stir for 1 hour at room temperature and 6 hours at 60°C. At that time, the pH is maintained at 6.8 to 7.0. This is cooled to room temperature, brought to pH 5.0 using 2N hydrochloric acid, and extracted three times with 50 ml each of acetic ester. Remove the acetate residue from the aqueous phase, cool to 0°C and PH2.0
acidify to. This is filtered with suction and allowed to dry. Yield: 2.4 g of the title compound. mp230℃
(Disassembly). NMR (DMSO- _d6 ): δ=8.1-7.5ppm (m, aromatic proton) δ=5.1ppm (d, 1H, C-7-H) δ=4.8ppm (d, 1H, C-6- H) δ=4.3ppm (d, 2H, CH2 _- S) δ=3.6ppm (AB, 2H, C-2- _CH2 ) Example 5 7-β-phenylacetamide-3-[(4-nitro -3-carboxy-phenyl)-thiomethyl]-3-cephem-4-carboxylic acid 7-amino-3-[(4-nitro-3-carboxy-phenyl)-thiomethyl]-3-cephem-4
- 822 mg of carboxylic acid are brought into solution with 500 mg of sodium bicarbonate in 200 ml of water, 20 ml of acetone are added and cooled to 0-5° C. in an ice bath. 350 mg of phenacetyl chloride in 5 ml of acetone are added dropwise while stirring. After stirring for 30 minutes, a further 50 mg of phenacetyl chloride in 2 ml of acetone is added. After 30 minutes the acetone is removed and the aqueous phase is acidified to pH 2.0 using 2N hydrochloric acid. Collect the precipitate, wash with water and dry. 750 mg of the title compound are obtained, which is identical in physical properties to the compound obtained according to Example 2. Example 6 7-β-thien-2-yl-acetamide-3
-[(4-nitro-3-carboxy-phenyl)
-Thiomethyl]-3-cefem-4-carboxylic acid Example 5 Using a total of 415 mg of thien-2-yl-acel chloride in place of phenacetyl chloride
Operate according to the instructions. 670 mg of the title compound are isolated, which is identical in physical properties to the compound obtained according to Example 3.

Claims (1)

[Claims] 1. General formula [wherein, R ¹
represents hydrogen, [formula] or [formula], and X represents the formula (In the formula, R ² represents nitro and R ³ represents hydrogen or carboxy). 2 formulas (wherein Z is formyloxy, acetoxy, acetoacetoxy or aminocarbonyloxy, and R ¹ represents hydrogen, [formula] or [formula]) can be expressed as (In the formula, R ² represents nitro and R ³ represents hydrogen or carboxy)]
and, when R ¹ means hydrogen, optionally with [Formula] or [Formula] or a reactive derivative thereof,
general formula A method for producing a cephalosporin having the formula (wherein R ¹ and X have the above-mentioned meanings). 3 Formula H-X [wherein X is the formula (In the formula, R ² represents nitro and R ³ represents hydrogen or carboxy)]
The method according to claim 2, wherein a compound having the following is used in the form of a salt thereof. 4 General formula [In the formula, R ¹ represents hydrogen, [formula] or [formula], and X represents the formula (In the formula, R ² represents nitro and R ³ represents hydrogen or carboxy)]
β characterized in that the chromogen cephalosporin having
-Preparation for lactamase detection. 5. The formulation according to claim 4, wherein the carrier material is paper (test strip) or other suitable material.